Protein ubiquitination is a widespread mechanism for cellular regulation, and new regulators are valuable research tools and may help to generate therapeutic small molecules. Ernst et al. used known crystal structures to roughly define the interaction domain between a ubiquitin-specific protease and a ubiquitinated substrate and then screened ubiquitin variants with changes in these residues to find variants that acted as potent and specific regulators that could modify ubiquitin pathway regulation in cells.